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bioRxiv · 10.1101/2021.03.31.437818

A mechanistic basis for understanding the dual activities of the bifunctional Azotobacter vinelandii mannuronan C-5 epimerase and alginate lyase AlgE7

Gaardlos, M.·Heggeset, T. M. B.·Tondervik, A.·Teze, D.·Svensson, B.·Ertesvag, H.·Sletta, H.·Aachmann, F. L.
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Abstract

The functional properties of alginates are dictated by the monomer composition and molecular weight distribution. Mannuronan C-5 epimerases determine the former by epimerizing {beta}-D-mannuronic acid residues (M) into -L-guluronic acid residues (G). The molecular weight is affected by alginate lyases, which cleave alginate chains through {beta}-elimination. The reaction mechanisms for the epimerization and cleavage are similar and some enzymes can perform both. These dualistic enzymes share high sequence identity with mannuronan C-5 epimerases without lyase activity, and the mechanism behind their activity as well as the amino acids responsible for it are still unknown. In this study, we investigate mechanistic determinants of the bifunctional epimerase and lyase activity of AlgE7 from Azotobacter vinelandii. Based on sequence analyses, a range of AlgE7 variants were constructed and subjected to activity assays and product characterization by NMR. Our results show that the lyase activity of AlgE7 is regulated by the type of ion present: Calcium promotes it, whereas NaCl reduces it. By using defined poly-M and poly-MG substrates, the preferred cleavage sites of AlgE7 were found to be M{downarrow}XM and G{downarrow}XM, where X can be either M or G. By studying AlgE7 mutants, R148 was identified as an important residue for the lyase activity, and the point mutant R148G resulted in an enzyme with only epimerase activity. Based on the results obtained in the present study we suggest a unified catalytic reaction mechanism for both epimerase and lyase activities where H154 functions as the catalytic base and Y149 as the catalytic acid. ImportancePost-harvest valorisation and upgrading of algal constituents is a promising strategy in the development of a sustainable bioeconomy based on algal biomass. In this respect, alginate epimerases and lyases are valuable enzymes for tailoring of the functional properties of alginate, a polysaccharide extracted from brown seaweed with numerous applications in food, medicine and material industries. By providing a better understanding of the reaction mechanism and of how the two enzyme reactions can be altered by changes in reaction conditions, this study opens for further applications of bacterial epimerases and lyases in enzymatic tailoring of alginate polymers.

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Gaardlos, M., Heggeset, T. M. B., Tondervik, A., Teze, D., Svensson, B., Ertesvag, H., Sletta, H., Aachmann, F. L.. 2021-03-31. A mechanistic basis for understanding the dual activities of the bifunctional Azotobacter vinelandii mannuronan C-5 epimerase and alginate lyase AlgE7. https://doi.org/10.1101/2021.03.31.437818

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